This invention relates generally to novel mammalian brain cell types and methods of culturing such cells. The methods of the instant invention, which utilize suspension cultures and factors that limit cell contacts, result in an amplification of the production of neural stem and progenitor cells, and clones of such cells, from the adult mammalian brain, including the human brain and from tissue with significant (e.g. 1 day) postmortem intervals. Propagation of neural stem and progenitor cells is relevant to the large-scale production of glial and neuronal cells, and clones of such cells, as well as self-repair of the brain in neurological disease.
Prior to the present invention, cells from numerous tissues have been described that have attributes of stem or germ cells (i.e., spermatozoon or an ovum), and that are extremely well-suited for rapid self-renewal. Brain-derived stem cells have only recently been a major focus of attention, using a variety of lineage tracing and culture methodologies. See for example, Gage et al., Ann. Rev. Neurosci., 18:159-192 (1995); Svendsen et al., Trends Neurosci., 18:465-467 (1995); Alvarez-Buylla et al., Stem Cells, 13:263-272 (1995); Weiss et al., Trends Neurosci., 19:387-393 (1996); Steindler et al., Prog. Brain Res., 108:349-363 (1996); and Brustle et al., Neuron, 15:1275-1285 (1995).
Previous studies showed the presence of a dense extracellular matrix (xe2x80x9cECMxe2x80x9d) on and around subependymal zone (xe2x80x9cSEZxe2x80x9d) cells of the adult rodent (see, Gates et al., J. Comp. Neurol., 361:249-266 (1995); and Thomas et al., Glia, 17:1-14 (1996)). ECM molecules may facilitate cell movement and aspects of differentiation during development, and they are also implicated in a number of neuropathological conditions. Glycoproteins such as tenascin-C (TN) and proteoglycans such as the chondroitin sulfate-containing proteoglycans (CSPG) are expressed in high levels in the young brain, where they seem to have a role in forming glycoconjugate-rich boundaries around different functional groups of neurons, such as the somatosensory whisker barrel fields and striosomes in the striatum. They are then down-regulated in later stages of development (e.g. postnatal days 14-21) and normal adulthood, but their expression is enhanced in neuropathologic conditions, such as traumatic brain injury, where they are an important component of glial scar formation. In the astroglial/mesonchymal scar, they may create a barrier that inhibits the growth of neurites into the scar, although it has been proposed that some ECM molecules may actually encourage neuritic growth under some circumstances. It has also been suggested that ECM molecules regulate cell proliferation, differentiation, migration, and survival through cell-cell and cell-ECM interactions. Stem cells have been described in embryonic and postnatal mouse brain and in proliferative xe2x80x9cneurospheresxe2x80x9d that can be harvested and cultured from different brain areas, including the developing subventricular zone. See, for example, Cattaneo et al., Nature 347:762 (1990); Richards et al., Proc. Nat""l Acad. Sci. (USA), 89:8591-8595 (1992); Reynolds et al., Science, 255:1707-1710 (1992); Reynolds et al., J. Neurosci. 12:4565-4574 (1992); Reynolds et al., Dev. Biol., 175:1-13 (1996); Vescovi et al., Neuron, 11:951-966 (1993); Kirschenbaum et al., Cerebral Cortex, 6:576-589 (1994); Kirschenbaum et al., Proc Nat""l Acad. Sci. (USA), 92:210-214 (1995) Fillmore et al., Neurosci Abs., 21:1528 (1996); and Gritti et al., J. Neurosci., 16:1091-1100 (1996). Evidence from immunolabeling and cell birthday analyses has pointed to the existence of such cells in the adult SEZ. See, for example, Luskin et al., Neuron, 11:173-189 (1993); Menezes et al., J. Neurosci. 14:5399-5416 (1994); Levison et al., Neuron. 10:201-212 (1993); Gates et al., J. Comp. Neurol., 361:249-266 (1995), Zerlin et al., J. Neurosci. 15:7238 (1995); Thomas et al., Glia, 17:1-14 (1996); and Jankovski et al., J. Comp. Neurol., 371:376 (1996). The combination of stem/precursor cells, and a dense ECM in the peri-ventricular SEZ throughout the neuraxis has prompted the inventors of the instant invention to refer to this area as being the neuropoietic xe2x80x9cBrain Marrowxe2x80x9d (Steindler et al, Pros. Brain Res. 108: 349, (1996)) since it contains elements in common with hematopoietic bone marrow.
In addition, it has recently been described that small numbers of neurons were found to arise from precursor cells of adult human temporal lobe (Kirschenbaum et al., Cerebral Cortex, 6:576-589 (1994), Laywell et al., Neurosci. Abs. 23:297 (1997)). The production of proliferating progenitor cells from the adult rodent brain and spinal cord has also been recently described (see, Gritti et al., J. Neurosci., 16:1091-1100 (1996); and Weiss et al., J. Neurosci., 16,7599-7609 (1996)). This is surprising in that with few exceptions, neuronogenesis has traditionally been thought to end shortly after birth in the mammalian central nervous system (CNS) (see, Gage et al., Ann. Rev. Neurosci. 18:159-192 (1995)). The possibility that multipotential stem cells persist in the adult brain has implications for neuroregeneration and CNS transplantation. Accordingly, there is a need in the art for such technology. This need is met by the present invention.
The present invention discloses an advancement in the biological arts in which previously unknown brain stem cells are cultured and isolated. The brain stem cells are characterized in that the daughter cells of the brain stem cells differentiate into neurons and glia and, therefore, are useful in neuroregeneration cell biology, and CNS drug-effects and drug-discovery studies. The novel method of isolating such cells comprises culturing dissociated adult mammalian brain in conditions that affect cell-substrate and cell-cell contacts. The cultured aggregates survive transplantation to the adult mammalian brain. Following transplantation, the daughter cells of the transplanted stem cells differentiate into other cell types, including but not limited to glia, neurons, astrocytes, and oligodendrocytes, thus allowing for replacement of cells damaged by injury or disease.
Studies of the ECM molecules in the adult brain revealed the existence of an ECM-rich pathway within which neuronal progenitor cells proliferate and migrate. These ECM molecules play a significant role in these events. According to the invention, the in vitro manipulation of these and related molecules affects cellular adhesity to other cells or substrates, and affects the growth of neural stem and progenitor cells, as described below.
To discourage cell-cell interactions that induce cellular differentiation, and thus contribute to an increased cellular proliferation of stem/progenitor cells, dissociated cells from the adult brain were cultured in factors that interfere with protein-protein interactions, or in gelatinous organic substances that also discourage cell contacts and allow the isolation of clonally-related colonies (spheres) of cells. There is a great expansion of the numbers of adult brain stem and progenitor cells due to these conditions, with potentially up to millions of neuronal and glial progenitors from small numbers of founder cells in less than three months. When aggregates of progenitor cells are plated on particular extracellular matrix molecule substrates in the presence of different growth factors, hormones, steroids, and other factors (see, Examples 3, and 10), they differentiate into neurons and glial cells. Such cells are suitable for studies of drug-discovery and testing using clones of glial and neuronal cells as well as for cell replacement therapies in a variety of brain structures (e.g. in the brain or spinal cord for regeneration or space-occupation, as in spinal cord syrinx injuries, or stroke cavities, arteriovenous malformations, epileptic foci, or peripheral nerve neuromas).
Stem cells appear to make up 0.001-0.01% of an entire population of cells in renewing or potentially renewing tissues such as bone and xe2x80x9cbrain marrowxe2x80x9d (see, Thomas et al., Glia, 17:1-14 (1996)). Hence, any method that assures a large scale production of differentiated cells from a small number of the most primitive stem/precursor cells is extremely useful for self-repair (autologous cell replacement) therapies following traumatic or degenerative disease. The invention harvests brain cells from a variety of sources including, but not limited to, rodent brain specimens, human brain biopsy, and post-mortem mammalian brain tissue. The invention method includes isolation and amplification of neural cells as a therapeutic self-repair approach for neurological disease, particularly human disease. To accomplish the isolation and amplification, the present invention uses a novel tissue culture approach to maximize the isolation of stem cells, and to assure a maximal number of transplantable cells from a very small number of stem cells, thus assuring the least risk for complications from a targeted and small biopsy site. At the same time, the culture method offers the possibility for large scale cell production from a very limited number of extracted stem cells. Furthermore, since a more primitive type of stem/precursor cell, than any seen prior to now, has been isolated and expanded using the invention technology, this cell type is more amenable to central nervous system (CNS) transplantation and integration, as well as gene transduction approaches. For example, using various vectors, the isolated cells of this invention can be transduced with genes coding for desired functions. For example, vectors containing genes coding for adhesion molecules can be transduced into these cells to increase cellular adhesivity. This would be useful, for example, to produce glia with increased adhesivity for cavity-filling approaches. Alternatively, the isolated cells of the present invention can be transduced with genes coding for growth factors and directing neuronal phenotype, including neurotransmitter associated genes to produce neurons capable of increased neurotransmitter production. In addition, because the cells of the present invention are pluripotent, they are extremely well suited for cell-grafting approaches.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the invention as claimed. Other objects, advantages and novel features will be readily apparent to those skilled in the art from the following detailed description of the invention, when considered in conjunction with the accompanying drawings.